Composition and device for in vivo cartilagerepair

ABSTRACT

The composition as described serves for in vivo cartilage repair. It basically consists of a naturally derived osteoinductive and/or chondroinductive mixture of factors (e.g. derived from bone) or of a synthetic mimic of such a mixture combined with a nanosphere delivery system. A preferred mixture of factors is the combination of factors isolated from bone, known as BP and described by Poser and Benedict (WO 95/13767). The nanosphere delivery system consists of nanospheres defined as polymer particles of less than 1000 nm in diameter (whereby the majority of particles preferably ranges between 200-400 nm) in which nanospheres the combination of factors is encapsulated. The nanospheres are loaded with the mixture of factors in a weight ratio of 0.001 to 17% (w/w), preferably of 1 to 4% (w/w) and have a release profile with an initial burst of 10 to 20% of the total load over the first 24 hours and a long time release of at least 0.1 per day during at least seven following days. The nanospheres are composed of e.g. ((D,L)-lactic acid/glycolic acid)-copolymer (PLGA). The loaded nanospheres are e.g. made by phase inversion. The composition is advantageously utilized as a device comprising any biodegradable matrix in which the nanospheres loaded with the factor combination is contained.

BACKGROUND OF THE INVENTION

Articular cartilage, an avascular tissue found at the ends ofarticulating bones, has no natural capacity to heal. During normalcartilage ontogeny, mesenchymal stem cells condense to form areas ofhigh density and proceed through a series of developmental stages thatends in the mature chondrocyte. The final hyaline cartilage tissuecontains only chondrocytes that are surrounded by a matrix composed oftype II collagen, sulfated proteoglycans, and additional proteins. Thematrix is heterogenous in structure and consists of threemorphologically distinct zones: superficial, intermediate, and deep.Zones differ among collagen and proteoglycan distribution,calcification, orientation of collagen fibrils, and the positioning andalignment of chondrocytes (Archer et al., J. Anat. 189(1): 23-35, 1996;Morrison et al., J. Anat. 189(1): 9-22 1996, Mow et al., Biomaterials13(2): 67-97, 1992). These properties provide the unique mechanical andphysical parameters to hyaline cartilage tissue.

In 1965, a demineralized extraction from bovine long bones was found toinduce endochondral bone formation in the rat subcutaneous assay (UristScience 150: 893-899, 1965). Seven individual factors, termed BoneMorphogenetic Proteins (BMPs), were isolated to homogeneity and, becauseof significant sequence homology, classified as members of the TGFβsuper-family of proteins (Wozney, et al., Science 242: 1528-34, 1988;Wang et al., Proc. Nat. Acad. Sci. 87: 2220-2224, 1990). Theseindividual, recombinantly-produced factors also induce ectopic boneformation in the rat model (Luyten et al., J. Biol. Chem. 264: 13377-80,1989; Celeste et al., Proc. Nat. Acad. Sci. 87: 9843-50, 1990). Inaddition, in vitro tests have demonstrated that both BMP-2 and TGFβ-1induce mesenchymal stem cells to form cartilage (Denker, et al.,Differentiation 59(1): 25-34, 1995; Denker et al., 41st Ann. Orthop.Res. Society 465: 1995). Both BMP-7 and BMP-2 have been shown to enhancematrix production of chondrocytes in vitro (Flechtenmacher J. ArthritisRheum. 39(11): 1896-904, 1996: Sailor et al., J. Orthop. Res. 14:937-945, 1996). From these data we can conclude that not only are theBMPs important regulators of osteogenesis, but that they also playcrucial roles during chondrogenic development in vitro.

A partially-purified protein mixture from bovine long bones, termed BP(Bone Protein), also induces cartilage and bone formation in the ratsubcutaneous assay (Poser and Benedict, WO95/13767). BP in combinationwith calcium carbonate promotes bone formation in the body. In vitro, BPinduces mesenchymal stem cells to differentiate specifically to thecartilage lineage, in high yields, and to late stages of maturation(Atkinson et al., J. Cellular Biochem. 65: 325-339, 1997).

The molecular mechanism for cartilage and bone formation has beenpartially elucidated. Both BMP and TGFβ molecules bind to cell surfacereceptors (the BMP/TGFβ receptors), which initiates a cascade of signalsto the nucleus that promotes proliferation, differentiation tocartilage, and/or differentiation to bone (Massague Cell 85: 947-950,1996).

In 1984, Urist described a substantially pure, but not recombinant BMP,combined with a biodegradable polylactic acid polymer delivery systemfor bone repair (U.S. Pat. No. -4,563,489). This system blends togetherequal quantities of BMP and polylactic acid (PLA) powder (100 μg ofeach) and decreases the amount of BMP required to promote bone repair.

Hunziker (U.S. Pat. No. -5,368,858; U.S. Pat. No. -5,206,023) describesa cartilage repair composition consisting of a biodegradable matrix, aproliferation and/or chemotactic agent, and a transforming factor. A twostage approach is used where each component has a specific function overtime. First, a specific concentration of proliferation/chemotactic agentfills the defect with repair cells. Secondly, a larger transformingfactor concentration transforms repair cells into chondrocytes. Therebythe proliferation agent and the transforming agent may both be TGFβdiffering in concentration only. In addition, the patent discloses aliposome encapsulation method for delivering TFGβ-1 serving astransformation agent.

Hattersley et al. (WO 96/39170) disclose a two factor composition forinducing cartilaginous tissue formation using a cartilageformation-inducing protein and a cartilage maintenance inducing protein.Specific recombinant cartilage formation inducing protein(s) arespecified as BMP-13, MP-52, and BMP-12, and cartilagemaintenance-inducing protein(s) are specified as BMP-9. In oneembodiment, BMP-9 is encapsulated in a resorbable polymer system anddelivered to coincide with the presence of cartilage formation inducingprotein(s).

Laurencin et al., (U.S. Pat. No. -5,629,009) disclose achondrogenesis-inducing device, consisting of a polyanhydride andpolyorthoester, that delivers water soluble proteins derived fromdemineralized bone matrix, TGFβ, EGF, FGF, or PDGF.

The results of the approaches to cartilage repair as cited above areencouraging but they are not satisfactory. In particular, the repairtissue arrived at is not fully hyaline in appearance and/or it does notcontain the proper chondrocyte organization. Furthermore, previousapproaches to cartilage repair have been addressed to very small defectsand have not been able to solve problems associated with repair oflarge, clinically relevant defects.

One reason that previous approaches failed to adequately repaircartilage may be that they were not able to recapitulate naturalcartilage ontogeny faithfully enough, this natural ontogeny being basedon a very complicated system of different factors, factor combinationsand factor concentrations with temporal and local gradients. A singlerecombinant growth factor or two recombinant growth factors may lack theinductive complexity to mimic cartilage development to a sufficientdegree and/or the delivery systems used may not have been able to mimicthe gradient complexity of the natural system to a satisfactory degree.

Previous approaches may also have failed because growth factorconcentrations were not able to be maintained over a sufficient amountof time, which would prevent a full and permanent differentiation ofprecursor cells to chondrocytes. The loss of growth factor could becaused by diffusion, degradation, or by cellular internalization thatbypasses the BMP/TGFβ receptors. Maintaining a sufficient growth factorconcentration becomes particularly important in repair of large sizeddefects that may take several days or several weeks to fully repopulatewith cells.

The object of this invention is to create a composition for improvedcartilage repair in vivo. The inventive composition is to enable in vivoformation of repair cartilage tissue which tissue resembles endogenouscartilage (in the case of articular cartilage with its specificchondrocyte spatial organization and superficial, intermediate, and deepcartilage zones) more closely than repair tissue achieved using knowncompositions for inducing cartilage repair. A further object of theinvention is to create a device for cartilage repair which devicecontains the inventive composition.

This object is achieved by the composition and the device as defined bythe claims.

BRIEF DESCRIPTION OF THE INVENTION

The inventive composition basically consists of a naturally derivedosteo-inductive and/or chondroinductive mixture of factors (e.g. derivedfrom bone) or of a synthetic mimic of such a mixture combined with ananosphere delivery system. A preferred mixture of factors is thecombination of factors isolated from bone, known as BP and described byPoser and Benedict (WO 95/13767). The nanosphere delivery systemconsists of nanospheres defined as polymer particles of less than 1000nm in diameter (whereby the majority of particles preferably rangesbetween 200-400 nm) in which nanospheres the combination of factors isencapsulated. The nanospheres are loaded with the mixture of factors ina weight ratio of 0.001 to 17% (w/w), preferably of 1 to 4% (w/w) andhave an analytically defined release profile (see description regardingFIG. 2) showing an initial burst of 10 to 20% of the total load over thefirst 24 hours and a long time release of at least 0.1 per day during atleast seven following days, preferably of 0.1 to 1% over the following40 to 60 days. The nanospheres are composed of e.g. (lacticacid-glycolic acid)-copolymers (Poly-(D,L)lactic acid-glycolic acid)made of 20 to 80% lactic acid and 80 to 20% of glycolic acid, morepreferably of 50% lactic acid and 50% of glycolic acid.

The loaded nanospheres are e.g. made by phase inversion according toMathiowitz et al. (Nature, 386: 410-413, 1997) or by other methods knownto those skilled in the art (Landry, Ph.D Thesis, Frankfurt, Germany).

The inventive composition is advantageously utilized as a devicecomprising any biodegradable matrix including collagen type I and II,and hyaluronic acid in which matrix the nanospheres loaded with thefactor combination is contained. The matrix can be in the form of asponge, membrane, film or gel. The matrix should be easily digestible bymigrating cells, should be of a porous nature to enhance cell migration,and/or should be able to completely fill the defect area without anygaps.

It is surprisingly found that the inventive composition consisting of anosteo-inductive and/or chondroinductive combination of factors (e.g.derived from natural tissue) encapsulated in nanospheres as specifiedabove, if applied to a defect area of an articular cartilage, leads tothe transformation of virtually all precursor cells recruited to therepair area to chondrocytes, and furthermore, leads to a homogenouschondrocyte population of the repair area and to a chondrocyte order andanisotropic appearance as observed in endogenous hyaline cartilage.These findings encourage the prospect that the inventive composition maylead to significant improvements also regarding repair of large defects.

As mentioned above, instead of an osteoinductive and/or chondroinductivemixture of factors derived from bone (BP), the inventive composition maycomprise natural factor mixtures derived from other tissues (e.g.cartilage, tendon, meniscus or ligament) or may even be a syntheticmimic of such a mixture having an osteoinductive and/or chondroinductiveeffect. Effective mixtures isolated from natural tissue seem to containa combination of proliferation, differentiation, and spatial organizingproteins which in combination enhance the tissue rebuilding capacitymore effectively than single proteins (e.g. recombinant proteins).

The specified, analytically defined release profile of such factormixtures from nanospheres results in the formation of concentrationgradients of proliferation and differentiation factors, which obviouslymimics the complex gradients of factors observed during naturaldevelopment very well. The nanosphere extended release profile issufficient to provide growth factor during the time frame that repaircells arrive into the matrix. The release profile obviously leads to ahomogenous population of a matrix with precursor cells, to fulldifferentiation of virtually all of the precursor cells to chondrocytes,and to the formation of an endogenous hyaline cartilage structure.

Another advantage of the inventive composition is that when thenanospheres are placed in a matrix to form a device for cartilagerepair, they are randomly distributed and remain in place when in ajoint cartilage defect. During cellular infiltration anddifferentiation, the nanospheres are in the correct position over thecorrect time frame.

Nanospheres have been demonstrated to adhere to the gastrointestinalmucus and cellular linings after oral ingestion (Mathiowitz et al.,Nature, 386 410-413 1997). We envisage that nanospheres also adhere tocartilage precursor cells and furthermore, may also adhere to BMP/TGFβreceptors located on the cell membrane. This property allows localizedhigh-efficiency delivery to the target cells and/or receptors. Becauseof the nanosphere small size and the chemical properties, they are moreeffective than liposomes or diffusion controlled delivery systems. Theefficient delivery to the receptors will facilitate chondrogenesis.

Derived from the above findings, we envisage the following mechanism forcartilage repair using the effect of the inventive composition. Duringthe first 24 hours (initial burst) 10 to 20% of the total load of thefactor mixture is released from the nanospheres into the matrix anddiffuses into the synovial environment. Following the initial burst, thenanospheres begin to release factors at a slow rate, which producesgradients of proliferation, differentiation, and spatial organizingproteins. In response to such gradients, precursor cells migrate to thedefect site. The loaded nanospheres adhere to cartilage precursor cellsand to the BMP and TGFβ receptors to provide localized highly efficientdelivery. The precursor cells become differentiated to chondrocytes andsecrete type II collagen and cartilage-specific proteoglycans. Thecomposition of the present invention stimulates differentiation ofvirtually all of these cells to overt chondrocytes and induces anordered cartilage structure which closely resembles hyaline cartilage.Furthermore, we envisage that this release system will allow homogenousrepair of large defect sites and repair of defects from patients withlow quantities of precursor cells.

For in vivo cartilage repair, the inventive device consisting of amatrix and the loaded nanospheres is placed in a chondral lesion thatwas caused by trauma, arthritis, congenital, or other origin. The damagecan result in holes or crevices or can consist of soft, dying, or sickcartilage tissue that is removed surgically prior to implantation of thedevice. Because of the unique properties of the inventive deviceprecursor cells populate the matrix, differentiate to chondrocytes, andform hyaline cartilage.

Application of the inventive composition (without matrix) e.g. byinjection can be envisaged also, in particular in the case of smalldefects. Thereby at least 2 μg of the composition per ml of defect sizeis applied or at least 20 ng of the osteoinductive and/orchondroinductive mixture encapsulated in the nanospheres is applied perml defect size.

The inventive composition and the inventive device are suitable forrepair of cartilage tissue in general, in particular for articularcartilage and for meniscus cartilage.

BRIEF DESCRIPTION OF THE FIGURES

The following figures illustrate the physical and chemical parameters ofthe inventive composition, the in vitro cartilage inductive activity ofBP released from nanospheres and in vivo repair of an articularcartilage defect using the inventive device.

FIG. 1 shows a scanning electron micrograph of BP-loaded nanospheres;

FIG. 2 shows the release profile (cumulative release vs. time) of theinventive composition;

FIG. 3 shows the release profile of the inventive composition comparedwith release profiles of nanosphere delivery systems loaded with otherproteins;

FIG. 4 shows the volume of a cartilage defect vs. the days required forpopulating the defect with repair cells;

FIG. 5 shows micromass cultures in the presence or absence ofnanospheres loaded with BP;

FIG. 6 shows cartilage marker analyses for in vitro cultures containingBP only and for similar cultures containing the inventive composition;

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows a scanning electron micrograph of BP-loaded nanospheres.The microparticle sizes range from 100-1000 nm with the majority ofindividual particles ranging between 200-400 nm.

The release rate profile of the inventive composition was determined byin vitro analysis of BP delivered from nanospheres. These nanosphereswere made by phase inversion according to the method as disclosed byMathiowitz et al. (Nature 386, 410-414, 1997) of ((DL)lacticacid/glycolic acid)-copolymer containing the two acids in a weight ratioof 50:50 and they were loaded with 1% and with 4% of BP.

For determination of the release rate profile, the nanospheres wereplaced in a sterile saline solution and incubated at 37° C. BP releasedinto the supernatant was measured using a BCA assay (Pierce). BPreleased from the nanospheres as specified shows two successive anddistinct profile parts: a fast release (initial burst) of approximately10 to 20% of the loaded BP over the first 24 hours and a slow release of0.1 to 1% per day (cumulative 40% to 50%) over 40 to 60 days (FIG. 2).

The release is intermediate between zero-order and first-order kinetics.Both the 1% and 4% encapsulated BP have similar release profiles.

For attaining release rate profiles as specified above and as necessaryfor the improved results in cartilage repair the nanospheres are to beadapted accordingly when using factor mixtures other than BP. Thereby,e.g the composition of the nanosphere copolymer, the molecular weight ofthe polymer molecules and/or the loading percentage of the nanospheresmay be changed. The optimum nanosphere character for each specific casehas to be found experimentally whereby the release rate profile isanalyzed in vitro as described above.

In the same way, the nanosphere delivery system can be modifiedregarding the percentage of BP to be released in the first 24 hours,percentage of BP to be released after 24 hours and/or length of timeafter the first 24 hours during which the remainder of BP is released.In addition, the percentage of BP loaded to the nanospheres is of coursevariable too, whereby for obtaining the results as described for thespecified composition. all the modifications are to be chosen such thatthe resulting delivery keeps within the range as specified.

All of the above parameters can be modified to account for the patient'sage, sex, diet, defect location, amount of blood present in the defect,and other clinical factors to provide optimal cartilage repair. Forexample, nanospheres with longer release rates are used for treatinglarger defects and/or for patients with fewer precursor cells (e.g.older patients or patients with degenerative symptoms). In contrast,patients with larger quantities of progenitor cells and/or smallerdefects may require a shorter release rate profile.

FIG. 3 shows the release profile as shown in FIG. 2 for nanospheres asspecified above loaded with BP and with other proteins (same loadingpercentages) such as BSA (bovine serum albumin) or lysozyme. Thedrastically different release characteristics shows that the profile isdependent on the protein type also. Tne same is valid for a morehydrophobic mixture of bovine bone derived proteins (PIBP).

FIG. 3 illustrates the singularity of the inventive combinationconsisting of the specific delivery system (nanospheres as specifiedabove encapsulating the factors) and the specific protein mixture (BP)which is obviously the key to the improved results in cartilage repairas observed when using the inventive composition or device.

To determine the length of time required for precursor cell repopulationof different sized defects, the following calculation was performed. Weestimate that approximately 50,000 cells are recruited to thedefect/day. Since the cellular density of cartilage is about 4×10⁷cells/ml, a 10 μl volume defect will take approximately 8 days to fillwith cells. FIG. 4 plots the number of days required to fill differentvolume defects with cells. The Figure assumes an infinite supply ofcells and a constant rate of cell attraction to the defect site, Thegraph demonstrates that the larger a defect size is, the more time isrequired to completely fill it with cells. Since a 60 μl volume defectwill take over 45 days to fill, this Figure demonstrates the necessityfor a long term release of factors to induce differentiation of theprecursor cells over up to a two month period.

To determine whether BP bioactivity is harmed by the encapsulationprocess and to determine whether the released BP was fully bioactive,the following assay was performed. Previously, it was demonstrated that10T1/2 micromass cultures exposed to BP induce formation of a threedimensional spheroid structure that can be observed macroscopically intissue culture wells (Atkinson et al., J. Cellular Biochem. 65: 325-339,1997). BP concentrations equal or greater than 20 ng/ml were requiredfor spheroid formation. No spheroid forms in the absence of BP or atconcentrations less than 10 ng/ml (see following table). In this assay,10T1/2 mesenchymal stem cells act as in vitro models for the precursorcells recruited to a natural defect.

We employed the same assay to test the bioactivity of BP released from1% loaded nanospheres. BP was eluted from nanospheres at 37° C. in a 5%CO₂ humidified incubator. After 24 hours 16% BP is released; and between24 hours and 7 days, 7% BP was released (FIG. 2). The supernatant wascollected, serial dilutions were made, and the supernatant was added to10T1/2 micromass cultures. BP released from nanospheres at both timepoints formed spheroids at concentrations greater than 20 ng/ml, but notat concentrations between 0 and 10 ng/ml (see following table).Non-encapsulated BP also formed spheroids at concentrations greater than20 ng/ml, but not at concentrations between 0 and 10 ng/ml. We concludethat both nanosphere encapsulation and release of BP does not inhibit BPbioactivity.

Spheroid formation (−=no spheroid formation; +=spheroid formation): BPconcentration (ng/ml) state of used BP 0-10 20-1000 non-encapsulated BP− + released from nanospheres (24 h) − + released from nanospheres (168h) − +

To determine the effect of BP slow release in the direct presence ofmicromass cultures, the following assay was performed. Nanospheres werewashed for 24 hours and the supernatant was discarded. The nanosphereswere then added to micromass cultures at a quantity such that 10 or 25ng/ml of BP would be released over 24 hours. Release of 25 ng/mlresulted in spheroid formation whereas release of 10 ng/ml did not formspheroids (FIG. 5). Similarly, the addition of 10 ng of non-encapsulatedBP per ml did not form a spheroid whereas the addition of 25 ng ofnon-encapsulated BP per ml did form a spheroid. Regarding the specificin vitro set-up, we conclude that slow release of BP over 24 hours is aseffective as a single dose of BP.

To determine whether the BP released from nanospheres was aschondrogenic as non-encapsulated BP, spheroids were analyzed for type IIcollagen and proteoglycan content. 10T1/2 spheroids from the above assaythat had formed with 1 μg of released BP per ml or 1 μg ofnon-encapsulated BP per ml were tested histologically with Azure and H+Estains and immunocytochemically with antibodies to type II collagenafter 7 days. Both encapsulated and non-encapsulated BP inducedcartilage markers such as type II collagen, proteoglycan, and round cellshape (FIG. 6). In addition, no qualitative differences were observedbetween encapsulated and non-encapsulated BP with respect to cellquantity, viability, morphology, or organization (FIG. 6). We concludethat BP retains full chondrogenic capacity after release fromnanospheres.

The in vitro models used for determining the chondroinductive effect ofBP differ from the in vivo case by the fact that in the in vitro casethe precursor cells are present in an appropriate number and in anappropriate distribution whereas in the in vivo case the precursor cellsfirst have to populate the defect and for this reason have to migrateinto the defect. Only in the latter case and for achieving repaircartilage which resembles natural cartilage to a high degree, it isessential for the BP to be released over a prolonged time periodaccording to a specific release profile.

EXAMPLE

The following example shows that BP released from nanospheres inducescartilage repair in chondral defects in vivo whereby virtually all cellsrecruited to the defect become chondrocytes, whereby the cell structureobtained is ordered, and whereby a hyaline matrix is built up.

Using a sheep model, unilateral defects of 0.5 mm width, 0.5 mm depthand 8 to 10 mm length were created in the trochlear groove of thepatella. The defects did not penetrate the subchondral bone. The sheepemployed in this study were seven years old and displayed degenerativesymptoms, including brittle bones, chondromalacia, and subchondralcysts. Because of their advanced age and degenerative symptoms, theseamimals probably have decreased numbers of precursor cells. The defectswere then dressed according to Hunziker and Rosenberg (J. Bone JointSurg. 78A(5): 721-733, 1996) with minor changes. Briefly, afterenzymatic proteoglycan removal with Chondroitinase AC, 2.5 μl of asolution containig 200 units Thrombin per ml was placed in the defect.Then, a paste was filled into the defect, the paste containing per ml:60 mg Sheep Fibrinogen (Sigma), 88 mg Gelfoam (Upjohn) and either 10 μgof BP-nanospheres or 10 μg of BP-nanospheres plus 80 ng rhIGF-1 (R+DSystems).

The nanospheres used were the nanospheres as specified in thedescription regarding FIG. 2 and they were loaded with 1% (w/w) of BP.

Assuming that the in vitro determined release rate is approximately thesame as for the in vivo case, 10 to 20 ng BP per ml were released duringthe first 24 hours and approximately 0.1 to 1 ng per day for thefollowing approximately 60 days.

After eight weeks, necropsies were performed. The repaired cartilagehistology showed that virtually all of the precursor cells weredifferentiated to chondrocytes throughout the defect. In addition, therewas an ordered cartilage appearance with cells on the top being moreflattened morphologically than cells in the center and with the presenceof ordered, stacked chondrocytes in the lowest zone. The repairedcartilage was fully integrated into the endogenous tissue. In addition,the cartilage repaired with only BP-nanospheres was not significantlydifferent from the cartilage repaired using BP-nanospheres plus IGF-1.

In conclusion, these results demonstrate that BP released fromnanospheres is sufficient for cartilage repair and that no addintionalfactor is required (such as e.g recombinant factor IGF-1). Using theinventive device constitutes a one step method for cartilage repair,whereby the nanosphere release of BP is sufficient for differentiationof virtually all of the precursor cells to chondrocytes and forinduction of an ordered cartilage structure.

Other Publications:

Archer C W, Morrison E H, Bayliss M T, Ferguson M W: The development ofarticular cartilage: II. The spatial and temporal patterns ofglycosaminoglycans and small leucine-rich proteoglycans; J Anat(ENGLAND) 189 (Pt 1): 23-35 (1996)

Atkinson B L, Fantle, K S, Benedict J J, Huffer W E, Gutierrez-HartmannA: A Combination of Osteoinductive Bone Proteins DifferentiatesMesenchymal C3H/10T1/2 Cells Specifically to the Cartilage Lineage; J.Cellular Biochem. 65: 325-339 (1997).

Celeste A J, Iannazzi J A, Taylor R C, Hewick R M, Rosen V, Wang E A,Wozney J M: Identification of transforming growth factor beta familymembers present in bone-inductive protein purified from bovine bone;Proc Natl Acad Sci U S A, Dec, 87(24): 9843-7 (1990)

Denker A E, Nicoll S B, Tuan R S: Formation of cartilage-like spheroidsby micromass cultures of murine C3H10T1/2 cellls upon treatment withtransforming growth factor β1′; Differentiation 59(1): 25-34 (1995)

Denker A E, Nicoll S B, Tuan R S: 41st Annual Meeting Orthop. Res.Society. (abstract): 465 (1995)

Flechtenmacher J, Huch K, Thonar E J, Mollenhauer J A, Davies S R,Schmid T M, Puhl W, Sampath T K, Aydelotte M B, Kuettner K E:Recombinant human osteogenic protein 1 is a potent stimulator of thesynthesis of cartilage proteoglycans and collagens by human articularchondrocytes; Arthritis Rheum, Nov, 39(11): 1896-904 (1996)

Hunziker E B and Rosenberg L C: Repair of Partial-Thickness Defects inArticular Cartilage: Cell Recruitment from the Synovial Membrane; J.Bone Joint Surgery 78-A(5): 721-733 (1996)

Kim S, Turker M S, Chi E Y, Sela S, Martin G M: Preparation ofmultivesicular liposomes; Bioch. et Biophys. Acta 728:339-348 (1983)

Landry F B: Degradation of Poly (D,L-lactic acid) Nanoparticles inartificial gastric and intestinal fluids; in vivo uptake of thenanoparticles and their degradation products; Thesis for the Dept. ofBiochemistry, Pharmacy, and Food Chemistry of the Johann Wolfgang GoetheUniversity in Frankfurt, Germany

Luyten F P, Cunningham N S, Ma S, Muthukumaran N, Hammonds R G, Nevins WB, Woods W I, Reddi A H: Purification and partial amino acid sequence ofosteogenin, a protein initiating bone differentiation; J Biol Chem,264(23): 13377-80 (1989)

Massague J: TGFβ Signaling: Receptors, Transducer, and Mad Proteins;Cell 85: 947-950 (1996)

Mathiowitz E, Jacob J S, Jong Y S, Carino G P, Chickering D E,Chaturvedi P, Santos C A, Vijayaraghavan K, Montgomery S, Bassett M,Morrell C: Biologically erodable microspheres as potential oral drugdelivery systems; Nature 386: 410-4 (1997)

Morrison E H, Ferguson M W, Bayliss M T, Archer C W: The development ofarticular cartilage: I. The spatial and temporal patterns of collagentypes; J Anat (ENGLAND) 189(Pt 1): 9-22 (1996)

Mow V C, Ratcliff A, Poole A R: Cartilage and diarthrodial joints asparadigms for hierarchical materials and stuctures; Biomaterials 13(2):67-97 (1992) Sailor L Z, Hewick R M, Morris E A: Recombinant human bonemorphogenetic Protein-2 maintains the articular chondrocyte phenotype inlong-term culture; J. Orthop. Res. 14: 937-945 (1996)

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Wang E A, Rosen V, D'Alessandro J S, Bauduy M, Cordes P, Harada T,Israel D I, Hewick R M, Kerns K M, LaPan P, Luxenberg D P, McQuaid D,Moutsatsos I, Nove J, Wozney J M: Recombinant human bone morphogeneticprotein induces bone formation; Proc Natl Acad Sci U S A, 87(6): 2220-4(1990)

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1-23. (canceled)
 24. A composition comprising: a chondroinductiveprotein mixture; and a delivery system having an initial first releaserate (% total protein mixture load/time) greater than a subsequentsecond release rate (% total protein mixture load/time).
 25. Thecomposition of claim 1, wherein the first rate is greater than 10% perday.
 26. The composition of claim 1, wherein the second rate is lessthan 1% per day.
 27. The composition of claim 1, wherein the deliverysystem has a third release rate.
 28. The composition of claim 4, whereinthe third release rate is greater than 0.1% per day.
 29. The compositionof claim 4, wherein the third release rate occurs temporally before thesecond release rate.
 30. The composition of claim 1, wherein the proteinmixture comprises a bone derived protein.
 31. The composition of claim1, wherein the delivery system comprises nanospheres.
 32. Thecomposition of claim 1, further comprising a matrix.
 33. The compositionof claim 9, wherein the matrix comprises collagen.
 34. A compositioncomprising: a chondroinductive protein mixture; and a delivery systemhaving an initial first release rate greater than 10% total proteinmixture load per day and a subsequent second release rate less than 1%total protein mixture load per day.
 35. The composition of claim 10,wherein the delivery system has a third release rate.
 36. Thecomposition of claim 12, wherein the third release rate is greater than0.1% per day.
 37. The composition of claim 12, wherein the third releaserate occurs temporally before the second release rate.
 38. Thecomposition of claim 10, wherein the protein mixture comprises a bonederived protein.
 39. The composition of claim 10, wherein the deliverysystem comprises nanospheres.
 40. The composition of claim 10, furthercomprising a matrix.
 41. The composition of claim 17, wherein the matrixcomprises a material selected from the group consisting of type Icollagen, type II collagen and hyaluronic acid.
 42. The composition ofclaim 17, wherein the matrix comprises a material selected from thegroup consisting of type I collagen.
 43. A composition comprising: abone derived chondroinductive protein mixture; a biodegradable matrixincluding collagen; and a nanosphere delivery system having an initialfirst release rate greater than 10% total protein mixture load per dayand a subsequent second release rate less than 1% total protein mixtureload per day.